In vitro model of macrosteatotic (fatty) liver

ABSTRACT

The present invention relates to a system and methods for identifying a compound for de-fatting and functional recovery of macrosteatotic hepatocytes.

CROSS REFERENCE TO RELATED APPLICATION

This application claims priority of U.S. Provisional Application No.61/759,113 filed on Jan. 31, 2013. The content of the application isincorporated herein by reference in its entirety.

GOVERNMENT INTERESTS

The invention disclosed herein was made, at least in part, withGovernment support under Grant No. R01DK059766 and UH2 NS080733 from theNational Institutes of Health. Accordingly, the U.S. Government hascertain rights in this invention.

FIELD OF THE INVENTION

This invention relates to a system and methods for identifying compoundsor agents for de-fatting and functional recovery of macrosteatotichepatocytes. This system enables identifying key hepatic intracellularpathways, which govern the pathology of non-alcoholic fatty liverdisease.

BACKGROUND OF THE INVENTION

The liver is a vital organ in various vertebrates and some otheranimals. In the human body, the liver is the largest internal organ,providing many essential functions, including metabolic, exocrine andendocrine functions. The liver is necessary for survival as withoutliver function a human can only survive up to 24 hours. Disorders of theliver, including liver failure and end-stage liver diseases, areresponsible for a large number of deaths around the world and are amajor burden on the health care system. Based on the organ procurementand transplantation network (OPTN), transplantation of whole liver fromcadaveric donors was shown to be effective in saving the lives of about7,000 patients who receive such livers annually in the U.S. However,about 16,000 patients remain untreated on the waiting list annually.This shortage of suitable livers leads to about 4,000 death in the USalone.

A common reason of liver donor ineligibility is excessive fat content,known as macrosteatosis, which is characterized by the presence of largelipid droplets inside the parenchymal cells of the liver, hepatocytes.Furthermore, these droplets displace the nuclei to the cell periphery.Methods to recover such livers could enhance donor availability. Onesuch method that has been proposed is to subject these livers to machineperfusion in the presence of compounds that promote accelerated lipiddroplet breakdown and metabolism. However, a thorough exploration ofsuch compounds, and combinations thereof, is necessary to achieve“de-fatting” of the livers in a timescale of a few hours.

Thus, there is a need for systems and methods for identifying compoundsor agents for de-fatting and functional recovery of macrosteatotichepatocytes.

SUMMARY OF INVENTION

This invention relates to a system and methods for identifying compoundsor agents for de-fatting and functional recovery of macrosteatotichepatocytes.

In one aspect, the invention provides an in vitro culture system havinga cell population containing cultured or isolated macrosteatotichepatocytes and a culture medium. In one embodiment, the culture systemis an in vitro co-culture system that includes macrosteatotichepatocytes and additional cells, such as fibroblasts, normalhepatocytes, Kupffer cells, other combinations of livers cells, or cellsfrom non-liver organs.

Preferably, a substantial portion (e.g., more than about 50, 60, 70, 80,90, 95, or 99%) of the population are macrosteatotic hepatocytes. Themacrosteatotic hepatocytes can be maintained in a collagen matrix suchas a collagen matrix in a sandwich configuration and other matrixes suchas Matrigel™. The macrosteatotic hepatocytes can be derived from ananimal, including a human or a non-human mammal, such as a rodent (e.g.,a rat or mouse).

In the culture system, the macrosteatotic hepatocytes can contain lipidmacrodroplets or contain a higher (e.g., about 2, 3 or 4 times) amountof triglyceride as compared to control normal hepatocytes. The culturemedium can be one selected from the group consisting of a standardmedium, a steatosis inducing medium, and a steatosis reducing medium asdisclosed herein.

In a second aspect, the invention provides a screening method ofidentifying a compound or composition for de-fatting and functionalrecovery of macrosteatotic hepatocytes. The method includes thefollowing steps: obtaining a first culture system described above, whichhas a population of cultured macrosteatotic hepatocytes; incubating themacrosteatotic hepatocytes in a test medium containing a test compoundor test composition for a first period of time; and determining amacrosteatosis level or a function level of the macrosteatotichepatocytes. The test compound is determined as being effective forde-fatting and functional recovery of macrosteatotic hepatocytes if (i)the macrosteatosis level is lower than a control macrosteatosis level(e.g., by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or more) or (ii)the function level is higher than a control function level (e.g., byabout 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or more), or both. Thecontrol macrosteatosis or function level can be determined in the samemanner from a second culture system obtained and incubated in the samemanner as the first culture system except that the second system isincubated in a control medium free of the test compound. In oneembodiment, the macrosteatosis level can be selected from the groupconsisting of a level of lipid macrodroplets (e.g., sizes of lipiddroplets or an average thereof) and a level of triglyceride as well assecreted products indicating defatting (e.g., secreted triglyceride inthe form of very low density lipoprotein, VLDL or secreted ketonemolecules which indicate hepatic fat oxidation). In addition,macrosteatotic hepatocytes are defined as containing large lipiddroplets that can displace the nucleus to the periphery of thehepatocyte cytoplasm. Therefore, reversing this nucleus displacement bythe lipid droplets following defatting, can be an indicator ofmacrosteatosis defatting. The functional level can be selected from thegroup consisting of a level of urea secretion (e.g., urea secretion ratein terms of μg/10⁶ cells/day) and a level of bile canalicular function,which can be determined by e.g., examining carboxy-DCFDA accumulationand morphology as described below. In the method, the first period oftime can be about 2 hours to about 3 days, such as 2 days. The methodcan further include, after the first period of time, culturing themacrosteatotic hepatocytes in a standard medium for a second period oftime. The second period of time can be about 2 hours to about 3 days(e.g., about 2 days). The above-mentioned time scale of days in a staticculture system is equivalent to only several hours in a perfusion system(Nagrath et al. Metabolic Engineering. 11(4-5):274-83).

In yet another aspect, the invention features a method of producing apopulation of cultured macrosteatotic hepatocytes described above. Themethod includes providing a population of hepatocytes; culturing thepopulation of hepatocytes on a collagen matrix for a first period oftime; distributing a collagen gel solution over the hepatocytes tocreate a collagen sandwich configuration so that the hepatocytes are inthe middle of the collagen sandwich configuration; culturing thehepatocytes in the collagen sandwich configuration for a second periodof time; culturing the hepatocytes in a steatosis inducing medium for athird period of time to obtain a population of cultured macrosteatotichepatocytes. The first period of time can be about 12 hours to 48 hours(e.g., about 18-30 hours or about 24 hours). The second period of timecan be about 2 days to 3 weeks (e.g., about 3-5 days or about 4 days).The third period of time can be about 3-9 days, e.g., about 5-7 days orabout 6 days, depending on the concentration of the steatosis inducingagent in the medium.

In another aspect, the invention enables exploring key regulatorypathways in the pathological state of non-alcoholic fatty liver disease(NAFLD), which is characterized by macrosteatosis. This can be used toidentify lead to therapies to reverse NAFLD by de-fatting the livers andprevent its progression to nonalcoholic steatohepatitis (NASH).

The details of one or more embodiments of the invention are set forth inthe description below. Other features, objectives, and advantages of theinvention will be apparent from the description and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-E are diagram and photographs showing macrosteatosis inductionin primary rat hepatocytes: A. Experimental timeline. B. Hepatocytemorphology post-free fatty acids (FFA)-supplemented culture.Bright-field (top) and fluorescent images of Nile red and Hoechststained hepatocytes (bottom) for lean (day 8), microsteatotic (day 8),and macrosteatotic hepatocytes (day 11). White arrows=lipid droplets andcross-sectional areas. Red arrows=hepatocyte nuclei. C, D. IntracellularTG content and macrosteatotic (>350 μm²) droplet number as a function ofsteatosis. Means±S.E. N=6. *p<0.002 vs. D8 Lean, ⁺p<0.007 vs. D8 Fat. E.H&E-stained human steatotic liver. Arrows=macrovesicular lipid dropletsand cross-sectional surface areas. Bars=50 μm.

FIGS. 2A-D are a set of diagrams and photographs showing hepatocytemorphology and lipid content during macrosteatosis reduction.Macrosteatotic hepatocytes cultures were supplemented (SRS) or not(NSRS) with steatosis reducing agents for 2 days. A. Bright-field (top)and fluorescent images of Nile red and Hoechst stained macrosteatotichepatocytes (bottom) after 2 days of culture. B, C. Macrosteatotic (>350μm²) droplet number and intracellular TG content for different steatosisreduction media. Means±S.E. N=6. •p<0.01 vs. D13 Lean. ⁰p<0.03 vs. D11Fat. D. Bright-field images of initially SRS or NSRS treatedmacrosteatotic hepatocytes after 2 additional days of culture in NSRSmedium compared to lean controls. Bar=50 μm.

FIGS. 3A-C are a set of diagrams and photographs showing effect ofsteatosis induction and reversal on hepatocyte viability, albumin andurea secretion. Macrosteatotic hepatocytes were cultured in steatosisreducing medium until day 13 and then standard medium until day 15. A.Hepatocyte viability at the end of the steatosis reduction period (15days post-seeding). Note that nonviable hepatocytes (EtHD-1⁺) did notreduce steatosis. White arrows=EtHD-1⁺ cells. Bar=50 μm. B, C. Albuminand urea secretion rates normalized to viable cell number. Means±S.E.N=6. *p<0.0001 vs. day 5 lean and ⁺p<0.01 vs. lean at the same timepoint.

FIGS. 4A-D are a set of diagrams and photographs showing effect ofsteatosis induction and reversal on bile canaliculi morphology andfunction: A. Bile canalicular structures visualized by phase contrastappear as bright white hepatocyte borders (white arrows). B.Bright-field (top) and fluorescent images (bottom) of hepatocytesstained with green carboxy-DCFDA, which accumulates in functional bilecanaliculi, and blue Hoechst, which stains nuclei. Bar=50 μm. C, D.Esterase activity, assessed by the accumulation of calcein incalcein-AM-incubated cells, is not significantly different between leanand macrosteatotic hepatocytes. Data shown are means±S.E. N=5.

FIG. 5 is a set of photographs showing viability distribution inhepatocyte cultures after microsteatosis and macrosteatosis induction.Viable cell nuclei are stained with Hoechst (blue) but not EtHD-1 (red).Lean controls and methanol fixed cultures (which are all dead) are alsoshown for comparison. Bar=50 μm.

FIGS. 6A-B are bright-field (A) and H&E-staining (B) photographs showinglipid droplet minimal cross-sectional surface area definingmacrosteatosis. Lipid droplet sizes were measured within hepatocytecultures exposed to steatosis inducing medium for 6 days. A survey ofindividual cells revealed that nuclear dislocation to the cell peripheryoccurred when the cross-sectional surface area of lipid droplets reached350 μm2. Shown is a representative image of hepatocyte cultures showinglipid droplet sizes and nuclear location. Bar=50 μm.

FIGS. 7A-B are a set of photographs and a diagram showing morphologicalchanges of representative lipid droplets during macrosteatosisreduction: A. Bright-field time lapse images of macrosteatotichepatocytes during the first 36 h in SRS medium. Solid arrows=a singlemacrovesicular droplet; dashed arrows=nucleus in the same cell. Theimage sequence shows shrinking of the lipid droplet and return of thenucleus to the center. Bar=50 μm. B. Cross sectional surface area of thelipid droplet visualized in panel A decreases linearly with time.

FIG. 8 is a table showing supplemented amino acids supplemented tosteatosis reducing culture medium (SRS) and respective concentrations(mg/L).

FIGS. 9A-C are a set of photographs and a diagram showing effect of FFAconcentration on steatotic induction. Hepatocyte cultures were incubatedfor 6 days (Days 5-11 post-seeding with one media change at day 8post-seeding) in standard hepatocyte culture medium supplemented with 0,250, 500, 1000 or 2000 μM of oleic acid and the same concentration oflinoleic acid. A. Intrahepatic TG for these cultures on day 11post-seeding. *p<0.05 vs. 0 μM. B. Representative bright-field images(top), Nile red (green), and Hoechst (blue) stained cultures (bottom).Bar=50 μm. C. Hepatocyte percent viability in each of the cultures asassessed by EHD-1 assay. P=0.99 indicating no significant differenceamong the conditions. Means±S.E. N=5.

FIG. 10 is a photograph showing insulin induction-dependent AKTphosphorylation.

DETAILED DESCRIPTION OF THE INVENTION

This invention is based, at least in part, on unexpected discoveriesthat macrosteatotic hepatocytes can be generated and maintained in vitroand that steatosis reduction can be induced in vitro in culturedmacrosteatotic hepatocytes in a short term. It is also based on thediscoveries that macrosteatosis reversibly decreases hepatocyte functionand that supplementary agents accelerate macrosteatosis reduction andsome functional restoration with no deleterious effect on viability ofhepatocytes.

Accordingly, in one aspect, the invention provides a model system inwhich macrosteatosis can be induced by culturing hepatocytes in FFA-richmedium. This model is suitable to explore the effect of macrosteatosisreduction approaches on lipid droplet size as well as hepatocyteviability and liver-specific function. Macrosteatotic hepatocytecultures maintain high viability but decreased function. Acceleratedmacrosteatosis reduction does not adversely affect viability andaccelerates some functional restoration. This system is useful toimprove the understanding of the effect of macrosteatosis on lipidmetabolism and storage, liver-specific functions and hepatocellularresponse to stresses, such as ischemia/reperfusion (I/R) or its staticin vitro culture equivalent, hypoxia/reoxygenation (H/R).

This model can also serve as platform to screen and test various lipidmetabolism promoting agents that could eventually be used to reducemacrosteatosis in human livers. To that end, this model can be used inconjunction with other methods, such as I/R preconditioning and/oranti-oxidant-based methods to expand the liver donor pool (Nativ et al.,American Journal of Transplantation. 2012; Serafin et al., The AmericanJournal of Pathology. 2002; 161(2):587-601; Mokuno et al., LiverTransplantation. 2004; 10(2):264-72; and Vairetti et al. LiverTransplantation. 2009; 15(1):20-9).

In addition, this in vitro macrosteatotic hepatocyte model is useful toscreen agents for macrosteatotic reduction in donor livers beforetransplantation. As mentioned above, orthotopic liver transplantation isseverely limited by donor scarcity. This has motivated the developmentof strategies to recover livers from deceased donors currently notconsidered suitable for transplantation. Macrosteatosis, defined as theaccumulation of triglycerides (TG) in the form of large lipid dropletsthat displace the nucleus to the cell periphery, when found in more than30% of the hepatocytes, is a very common cause of donor ineligibility(de Graaf et al. J Gastroenterol Hepatol. 2012 March; 27(3):540-6 andSpitzer et al. Liver Transpl. 2010 July; 16(7):874-84). Such livers aremore sensitive to I/R injury inherent to liver transplantation, and moreprone to primary non-function, as well as increased morbidity andmortality post-transplantation. The incidence of hepatic macrosteatosisis likely to surge due to the obesity epidemic. Thus, techniques tosalvage macrosteatotic livers could significantly enhance donor supplyin both the short and long terms.

A variety of approaches targeting the downstream effects ofmacrosteatosis during I/R have shown promise in pre-clinical andclinical settings. However, several studies suggest that excessivehepatic lipid storage is a primary cause of the exuberant I/R response,especially when in the macrosteatotic form. Therefore, an alternativeapproach could be to eliminate the intracellular lipid droplets, thusdecreasing the frequency of macrosteatotic hepatocytes below acceptablelevels. Dieting, exercise, and fibrate drugs over several weeks havebeen shown to decrease macrosteatosis and enable living donor livertransplantation. In a rat liver model of macrosteatosis (induced by acholine and methionine-deficient diet; CMDD), switching to a normal diet3 days prior to transplantation reduced intrahepatic TG content by 35%and increased recipient viability from 0% to >75% post-transplantation(Berthiaume et al., Journal of Surgical Research. 2009; 152(1):54-60).

Diet/drug-induced macrosteatosis reduction occurs over days to weeks, atimescale that is not applicable to deceased human donors, which wouldrequire macrosteatotic reduction ex vivo within a few hours. Animalstudies have demonstrated the feasibility of macrosteatosis reductionvia machine perfusion of explanted steatotic livers. This process wasaccelerated by introducing agents that promote lipid metabolism(Jamieson et al. Transplantation. 2011; 92(3):289-95; Nagrath et al.Metabolic Engineering. 11(4-5):274-83; and Vairetti et al. LiverTransplantation. 2009; 15(1):20-9). However, a thorough exploration ofsuch agents and combinations thereof, has yet to be performed.Furthermore, there has been little investigation of the impact ofaccelerated macrosteatosis reduction on the viability and function ofhepatocytes, parameters that are critical for the successful outcome ofliver transplantation.

The cell culture model disclosed herein can be used to facilitate theevaluation of these agents with the ultimate goal of developingprotocols to promote accelerated macrosteatosis reduction and functionalrecovery of transplanted steatotic livers. Microsteatotic hepatocyteculture systems have been described in this context in the literature;however, their relevance is unclear given that clinical evidencesuggests that macrosteatotic—and not microsteatotic—livers arehypersensitive to I/R. As disclosed herein, a novel macrosteatotichepatocyte culture system is provided to investigate the effect ofmacrosteatosis on viability and liver-specific functions in hepatocytes.

As shown in the examples below, this system was successfully used toexplore the impact of accelerated macrosteatosis reduction on viabilityand the recovery of such functions. More specifically, a novel in vitroprimary rat macrosteatotic hepatocyte system was characterized wherebyintracellular TG accumulation and macrosteatotic lipid droplet formationwere induced by incubation in FFA-rich medium. It was found thatmacrosteatosis induction did not decrease hepatocyte viability, butsignificantly decreased urea secretion, disrupted the bile canalicularnetwork, and maintained albumin secretion rates below lean controls.Macrosteatosis was morphologically reversed by switching hepatocytesback to NSRS medium within, e.g. 4 days. Comparable macrosteatosisreduction was achieved in only 2 days with SRS medium. Hepatocyteviability remained high and did not depend on the macrosteatosisreduction rate. Macrosteatosis reduction led to the recovery ofliver-specific functions at different rates. Using the SRS medium, ureasecretion and the bile canalicular network fully recovered, whilealbumin secretion rate remained flat and below control lean hepatocytelevels during the macrosteatosis reduction experimental 4-day timeframe.Since some aspects of hepatocyte functional recovery were improved byaccelerated macrosteatosis reduction, this recovery may be dependentupon macosteatosis reduction time and/or may be sensitive to thecomposition of the supplements and their target pathways.

The study disclosed herein indicates that macrosteatosis significantlydecreased bile canalicular function, consistent with observations madepost 70% partial hepatectomy in a CMDD rat macrosteatotic liver model,where the recovery of the bile canalicular network was significantlydelayed. During recovery, the animals were given a normal diet, which isexpected to reduce the steatosis level to that of lean animals(Berthiaume et al., Journal of Surgical Research. 2009; 152(1):54-60;and Ninomiya et al., Transplantation. 2004; 77(3):373-9). In addition,steatotic rat liver perfusion studies indicate an increased bilesecretion rate post-steatosis reduction (Nagrath et al. MetabolicEngineering. 11(4-5):274-83; Vairetti et al. Liver Transplantation.2009; 15(1):20-9). Therefore, the steatosis reduction process could havecontributed to some aspects of functional recovery of macrosteatotichepatocytes.

As disclosed herein, addition of SRS significantly reduced lipid dropletsize compared to NSRS, but TG content was reduced to the same levelusing either media composition. This contrasts with prior studies usingmicrosteatotic hepatocyte cultures, where SRS promoted a higher degreeof TG reduction compared to NSRS medium (Nagrath et al. MetabolicEngineering. 11(4-5):274-83). The macrosteatotic culture system, whichcontains 2-fold higher TG levels compared to the microsteatotic system,may have impaired ability to eliminate lipolysis products from thecytoplasm, allowing their re-esterification to TG stored in small lipiddroplets, which may not be detectable using the Nile red stain method(Nagrath et al. Metabolic Engineering. 11(4-5):274-83; and 22. Gibbonset al., Biochem J. 1992; 284(Pt 2):457-62. If this is the case, furtherimprovement in TG removal can be achieved by preventing TGre-esterification, which would also help elucidate whether theredistribution of TG from macro- to micro-droplets, or its removal fromthe cell altogether, is required (Nir et al., American Journal ofTransplantation. 2012).

The above-described accelerated macrosteatosis reduction strategies canbe used and consequently recover some liver-specific function. Themacrosteatosis reduction time scale was ˜48 h to reduce the number ofmacrodroplets by ˜80%. In order to clinically translate this approach toliver grafts, this would need to be accomplished in a few hours.Interestingly, one study showed that ex vivo perfusion of obese Zuckerrat steatotic liver with a similar cocktail could achieve a significantreduction in lipid droplet size as well as 50% TG reduction within 3 h(Nagrath et al. Metabolic Engineering. 11(4-5):274-83; and Maguire etal., Biotechnology and Bioengineering. 2006; 93(3):581-91). Thus, it ispossible that faster dynamics occur in perfused livers, possibly due tothe flow conditions, which likely enhance nutrient and waste transportbetween cells and the bathing medium (Nir et al., American Journal ofTransplantation. 2012). To further improve the macrosteatotic hepatocyteculture model, one can introduce a flow component, thus enabling a morerigorous analysis of the combined effects of lipid metabolism promotingagents and flow parameters on the macrosteatosis reduction process (Niret al., American Journal of Transplantation. 2012).

As disclosed herein, the invention provides an in vitro model for livermacrosteatosis. This system can be used to screen defatting agents forfat cells, adipocytes or to identify improved diets or drugs to reducebody fat content in the contexts of obesity. This system contains cellsderived from starting cells, such as hepatocytes, hepatocyte-like cells,or their progenitor cells known in the art. Hepatocyte-like cell refersto a cell displaying one or more properties that are characteristic ofmature, parenchymal hepatocytes. In general, a hepatocyte-like cell maydisplay at least one, two, three, four, five or more of the followingproperties: ability to use pyruvate as a sole carbon source; phase Ibiotransformation capacity (e.g. ethoxyresorufin, pentoxyresorufin,testosterone); phase II biotransformation capacity (e.g. 1-chloro-2,4dinitrobenzene, 1,2-dichloro-4-nitrobenzene,7-chloro-4-nitrobenzene-2-oxa-1,3-diazole, estradiol, estrogen), thepresence of cytochrome P450 protein and gene expression; inducibility ofphase I and phase II biotransformation enzymes (e.g. β-naphthoflavone,phenobarbital, methylcholanthrene); albumin secretion, urea production,fibrinogen secretion, glycogen storage, the presence of the expressionof one or more of endogenous ALB, AFP, gamma-glutyryltransferase,hepatocyte nuclear factor (HNF) 1α, HNF 1β, HNF 3α, HNF 3β, HNF 4,HNF-6, anti-trypsin, CX32, MRP2, C/EBPα, transthyretin, CK-18 and/orCFTR; polygonal morphology.

Various cells from a subject or animal can be used as the startingcells. In some embodiments, the starting cells are stem cells. The stemcells useful for the method described herein include but not limited toembryonic stem cells, mesenchymal stem cells, bone-marrow derived stemcells, hematopoietic stem cells, chondrocyte progenitor cells, epidermalstem cells, gastrointestinal stem cells, neural stem cells, hepatic stemcells, adipose-derived mesenchymal stem cells, pancreatic progenitorcells, hair follicular stem cells, endothelial progenitor cells, andsmooth muscle progenitor cells. The stem cells can be pluripotent ormultipotent. In some embodiments, the stem cell is an adult, fetal orembryonic stem cell. The stem cells can be isolated from umbilical,placenta, amniotic fluid, chorion villi, blastocysts, bone marrow,adipose tissue, brain, peripheral blood, blood vessels, skeletal muscle,and skin. To convert stem cells to hepatocytes or hepatocyte-like cells,one needs to induce the stem cells so that they differentiate. Variousmethods were known in the art for achieving this purpose. See, e.g.,WO2011102532, WO2012058868, US20090191159, US20110070647, US2011004260,and US20120244129.

In a preferred example, starting cells are hepatocytes. To that end,hepatocytes can be isolated from a suitable animal (e.g., lean Zuckerrats) and maintained in a standard medium. To induce steatosis, thehepatocytes can then be cultured in a collagen sandwich and incubatedfor about 3-9 days (e.g., 5-7 days or 6 days) in a fattyacid-supplemented medium (i.e., a steatosis inducing medium). Thecultured cells can then be switched for 4 hours-6 days (e.g., 2 days) toa medium supplemented with lipid metabolism promoting agents (i.e.,steatosis reducing medium). During this period, intracellular lipiddroplet size distribution and triglyceride, viability, albumin and ureasecretion and bile canalicular function can be measured in the mannerdescribed in the example below or using methods known on the art.Generally, the fatty acid-supplemented medium should inducemicrosteatosis in about 3 days and macrosteatosis in about 6 days, withthe latter evidenced by large lipid droplets dislocating the nucleus tothe cell periphery.

As used herein, the term “a large lipid droplet” refers to anintracellular, triglyceride-containing droplet of a hepatocyte, wherethe area of such a droplet is about 200 μm²-2000 μm², for example 350 orabove, such as 350-2000 μm² as determined by a non-destructivequantitative image analysis method in the manner described in theexample section below.

As used herein, a standard medium or standard hepatocyte medium refersto any medium that can be used to culture or maintain hepatocyteswithout causing phenotype changes. Examples of the standard mediuminclude DMEM, C+H, Williams Medium E, HCM™ Hepatocyte Culture Medium,HMM™ Hepatocyte Maintenance Medium, InVitroGRO™ Hepatocyte Media andothers known in the art. A steatosis inducing medium is identical to astandard medium except that it contains one or more steatosis inductionagents, e.g., fatty acids such as oleic acid, linoleic acid, andpalmatic acid with bovine serum albumin (BSA) as fatty acid carrier,insulin and glucose at high concentrations, and heparinized plasma,which is high in free fatty acids. A steatosis reducing medium isidentical to a standard medium except that it contains one or moresteatosis reduction supplements. A reduction supplement or agent refersto any agent that increases lipid oxidation (for example mitochondrialbeta-oxidation) or lipid export (for example via secretion of VLDLparticles), such as forskolin, GW7647, scoparone, GW501516, hypericin,visfatin, and amino acids as well as bile acids.

The term “animal” includes all vertebrate animals including humans. Italso includes an individual animal in all stages of development,including embryonic and fetal stages. In particular, the term“vertebrate animal” includes, but not limited to, humans, non-humanprimates (particularly higher primates), canines (e.g., dogs), felines(e.g., cats); equines (e.g., horses), bovines (e.g., cattle), porcine(e.g., pigs), rodent (e.g., mouse or rat), guinea pig, cat, rabbit, aswell as in avians, such as birds, amphibians, reptiles, etc. The term“avian” refers to any species or subspecies of the taxonomic class ava,such as, but not limited to, chickens (breeders, broilers and layers),turkeys, ducks, a goose, a quail, pheasants, parrots, finches, hawks,crows and ratites including ostrich, emu and cassowary. Examples of anon-human animal include all non-human vertebrates, e.g., non-humanmammals and non-mammals mentioned above.

The invention provides a method of identifying a compound or acomposition for de-fatting and functional recovery of macrosteatotichepatocytes. The compound/composition thus-identified can be used topromote accelerated lipid droplet breakdown and metabolism in livertissue as well as reduce the cell's and or the graft's sensitivity toischemia/reperfusion injury associated with liver procurement andtransplantation.

Candidate compounds to be screened (e.g., proteins, peptides,peptidomimetics, peptoids, antibodies, small molecules, or other drugs)can be obtained using any of the numerous approaches in combinatoriallibrary methods known in the art. Such libraries include: peptidelibraries, peptoid libraries (libraries of molecules having thefunctionalities of peptides, but with a novel, non-peptide backbone thatis resistant to enzymatic degradation); spatially addressable parallelsolid phase or solution phase libraries; synthetic libraries obtained bydeconvolution or affinity chromatography selection; and the “one-beadone-compound” libraries. See, e.g., Zuckermann et al. 1994, J. Med.Chem. 37:2678-2685; and Lam, 1997, Anticancer Drug Des. 12:145. Examplesof methods for the synthesis of molecular libraries can be found in,e.g., DeWitt et al., 1993, PNAS USA 90:6909; Erb et al., 1994, PNAS USA91:11422; Zuckermann et al., 1994, J. Med. Chem. 37:2678; Cho et al.,1993, Science 261:1303; Carrell et al., 1994, Angew. Chem. Int. Ed.Engl. 33:2059; Carell et al., 1994, Angew. Chem. Int. Ed. Engl. 33:2061;and Gallop et al., 1994 J. Med. Chem. 37:1233. Libraries of compoundsmay be presented in solution (e.g., Houghten, 1992, Biotechniques13:412-421), or on beads (Lam, 1991, Nature 354:82-84), chips (Fodor,1993, Nature 364:555-556), bacteria (U.S. Pat. No. 5,223,409), spores(U.S. Pat. No. 5,223,409), plasmids (Cull et al., 1992, PNAS USA89:1865-1869), or phages (Scott and Smith 1990, Science 249:386-390;Devlin, 1990, Science 249:404-406; Cwirla et al., 1990, PNAS USA87:6378-6382; Felici 1991, J. Mol. Biol. 222:301-310; and U.S. Pat. No.5,223,409).

To identify a compound or composition mentioned above, one can contactor incubate a candidate compound or composition with the systemdisclosed herein. The cells can be macrosteatotic hepatocytes derivedfrom normal, lean hepatocytes as described in the example below. Afterthe incubation, one then measures macrosteatosis level or a functionlevel of the macrosteatotic hepatocytes. The level can be determined asdisclosed herein.

As disclosed herein, a number of ranges of values are provided. It isunderstood that each intervening value, to the tenth of the unit of thelower limit, unless the context clearly dictates otherwise, between theupper and lower limits of that range is also specifically disclosed.Each smaller range between any stated value or intervening value in astated range and any other stated or intervening value in that statedrange is encompassed within the invention. The upper and lower limits ofthese smaller ranges may independently be included or excluded in therange, and each range where either, neither, or both limits are includedin the smaller ranges is also encompassed within the invention, subjectto any specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

The term “about” generally refers to plus or minus 10% of the indicatednumber. For example, “about 20” may indicate a range of 18 to 22, and“about 1” may mean from 0.9-1.1. Other meanings of “about” may beapparent from the context, such as rounding off, so, for example “about1” may also mean from 0.5 to 1.4.

As used herein, the term “contacting” and its variants, when used inreference to any set of components, includes any process whereby thecomponents to be contacted are mixed into same mixture (for example, areadded into the same compartment or solution), and does not necessarilyrequire actual physical contact between the recited components. Therecited components can be contacted in any order or any combination (orsubcombination), and can include situations where one or some of therecited components are subsequently removed from the mixture, optionallyprior to addition of other recited components. For example, “contactingA with B and C” includes any and all of the following situations: (i) Ais mixed with C, then B is added to the mixture; (ii) A and B are mixedinto a mixture; B is removed from the mixture, and then C is added tothe mixture; and (iii) A is added to a mixture of B and C. “Contacting atemplate with a reaction mixture” includes any or all of the followingsituations: (i) the template is contacted with a first component of thereaction mixture to create a mixture; then other components of thereaction mixture are added in any order or combination to the mixture;and (ii) the reaction mixture is fully formed prior to mixture with thetemplate.

EXAMPLE

Experimental Procedures

Hepatocyte Isolation and Culture

Male lean Zucker rats, (Charles River, Wilmington, Mass.) (310±20 g)were housed in a 12 h light-dark cycle and temperature-controlledenvironment (25° C.) with water and standard chow ad libitum. Allexperimental procedures followed National Research Council guidelinesand were approved by the Rutgers University Animal Care and FacilitiesCommittee. Hepatocytes were isolated using a two-step in situcollagenase perfusion technique (Berthiaume et al., Journal of SurgicalResearch. 2009; 152(1):54-60 and Nagrath et al. Metabolic Engineering.11(4-5):274-83). Viability was 90±4% as determined by trypan-blueexclusion (Nagrath et al. Metabolic Engineering. 11(4-5):274-83).Six-well culture plates (Beckton-Dickinson, Franklin Lakes, N.J.) werepretreated with 50 ug/ml rat type 1 collagen solution(Beckton-Dickinson) in 0.02M acetic acid (Sigma-Aldrich, St. Louis, Mo.)overnight at 4° C. and washed with phosphate buffered saline (PBS,Invitrogen, Grand Island, N.Y.). Freshly isolated hepatocytes weresuspended (10⁶ cells/ml) in standard hepatocyte medium and seeded (10⁶cells/well) as previously described (Nagrath et al. MetabolicEngineering. 11(4-5):274-83) (15). After incubating the cells at 37° C.in a 90% air/10% CO₂ atmosphere for 24 h, medium was removed and acollagen gelling solution (0.5 ml/well) was added to form a gel overlay(Nagrath et al. Metabolic Engineering. 11(4-5):274-83). Cultures weremaintained in standard hepatocyte medium for 4 days with a fresh mediumchange every other day. Spent medium was collected (FIG. 1A,experimental days 1-5) for analysis.

Steatosis Induction and Reversal

Five-day hepatocyte cultures were switched to steatosis-inducing medium.Standard hepatocyte medium was supplemented with 2 mM oleic acid, 2 mMlinoleic acid, and 4% (weight to volume) bovine serum albumin(Sigma-Aldrich) for 3 days, as previously described (Berthiaume et al.,Journal of Surgical Research. 2009; 152(1):54-60 and Nagrath et al.Metabolic Engineering. 11(4-5):274-83). Medium was replaced with freshsteatosis-inducing medium for another 3 days of steatosis induction andthe spent medium was collected (FIG. 1A, experimental days 5-11). Theseconcentrations of free fatty acids (FFA) were chosen based on a doseresponse study to determine the FFA dose required to inducemacrosteatosis, as indicated in FIG. 9. Following 6-day steatosisinduction (11 days post-seeding), the medium was replaced with freshhepatocyte medium with no steatosis reduction supplements (NSRS), orwith a combination of the following steatosis reduction supplements(SRS): 10 uM forskolin, 1 uM GW7647, 10 uM scoparone (Sigma-Aldrich), 1uM GW501516, 10 uM hypericin (Enzo, Farmingdale, N.Y.), 0.4 ng/mlvisfatin (Biovision, Mountain view, Calif.) and amino acids (Invitrogen)at final concentrations described in FIG. 8. This cocktail promoted invitro microsteatosis reduction by activating hepatocellular TGmetabolism (Nagrath et al. Metabolic Engineering. 11(4-5):274-83). SRSmedium pH was adjusted to match that of NSRS. Cells were incubated inSRS or NSRS medium for 48 h, after which (13 days post-seeding) thespent medium from all cultures was collected and replaced with NSRSmedium for another 48 h. On post-seeding day 15, the spent medium fromall experimental conditions was collected (FIG. 1A).

Hepatocyte Steatosis Assessment

A non-destructive quantitative image analysis method was used toquantify lipid droplet size distribution. Hepatocyte cultures were fixedin 4% paraformaldehyde, stained with the lipid-specific Nile red stain(Adipored™, Lonza, Walkersville, Md.), and counterstained with 1 ug/mlnuclei-specific Hoechst-33342 stain (Invitrogen), following themanufacturer recommendations. Confocal fluorescence images were obtainedwith an Olympus IX-80 microscope and analyzed using an in-housealgorithm for edge detection, yielding unbiased measurements of size(cross sectional surface area) and lipid droplet distribution/cell. Inaddition, hepatocytes were scraped and sonicated in NSRS medium andusing a lipase assay kit (Sigma-Aldrich), the TG content measured byquantification of liberated glycerol (Berthiaume et al., Journal ofSurgical Research. 2009; 152(1):54-60). Time lapse bright-field imagesof hepatocytes undergoing macrosteatotic reduction were acquired every15 minutes for 36 hours using a temperature and gas controlled (37° C.and 10% CO₂ balanced with air) Olympus IX-80 microscope stage. The sizeof individual lipid droplets was monitored using SLIDEBOOK™ software(Intelligent Imaging Innovations, Denver, Colo.) to calculate a rate ofchange in cross sectional surface area.

Hepatocyte Viability Assessment

Cells were washed with PBS, and dead cells quantified followingincubation with 4.29 ug/ml Ethidium Homodimer-1 (EthD-1, Invitrogen),and 1 ug/ml Hoechst-33342 (Invitrogen) to stain all cells, for 20 min at37° C. (Maguire et al., Biotechnology and Bioengineering. 2006;93(3):581-91). Five 20× epifluorescence images were obtained in 5separate wells/experimental condition. Cells containing EthD-1 labelednuclei were counted as dead, while those with Hoechst-stained nucleiwere counted as live (FIG. 5). Percentage of viable cells was determinedin each well and averaged for 5 wells/condition (Maguire et al.,Biotechnology and Bioengineering. 2006; 93(3):581-91).

Hepatocyte Function Assessment

Rat albumin was measured by enzyme-linked immunosorbent assay (Bethyllaboratories, Montgomery, Tex.) and urea nitrogen using a biochemicalassay (Stanbio), both using spent media samples and following themanufacturer's recommendations. Concentrations were normalized to thetime period between medium changes and the number of viable hepatocytesto convert into specific secretion rates. Bile canaliculi function wasassessed by visualization of excreted fluorescent products afterincubation with 5-(and-6)-carboxy-2′,7′-dichlorofluorescein diacetate(carboxy-DCFDA) (Sigma-Aldrich) at a final concentration of 2 uM. Thismolecule passively enters the cytoplasm of normal hepatocytes, whereesterases metabolize it to a fluorescent product excreted into bilecanaliculi (Tuschl et al., Chemico-Biological Interactions. 2009;181(1):124-37). The nuclei were counterstained using Hochest-33342 asdescribed above. Images were captured with an Olympus IX-80 microscope.General intracellular esterase activity was assessed usingcalcein-acetoxymethylester (calcein-AM, Invitrogen) at 5 μg/ml. Fiveepifluorescence images were captured using a 20× objective on an OlympusIX-80 microscope in 5 separate wells/experimental condition. Thefluorescence levels obtained in each image were quantified usingSLIDEBOOK™ software (Intelligent Imaging Innovations) and averaged forthe 5 wells/condition.

Inducible cytochrome P450 activity was assessed as measured by breakdownof resorufin derivatives per established method (Tuschl et al.,Chemico-Biological Interactions. 2009; 181(1):124-37) and found positiveactivity in all groups (data not shown). The insulin sensitivity wasinvestigated via insulin withdrawal followed by induction as follows.The standard hepatocyte medium, which contains high levels of insulin (5Units/ml, Sigma-Aldrich), was removed and the cultures were washed 3times in PBS to remove any insulin residue. Insulin-free basal DMEM(Invitrogen) was added to the cells for 4 hours. Then, the medium wasremoved and either fresh insulin-free DMEM or insulin-rich DMEM (5Units/ml, Sigma-Aldrich) was added to the cells for 30 minutes.Intrahepatocellular protein was extracted and analyzed forphosphorylated AKT (at Thr 308) as well as total AKT by Western blotusing the appropriate antibodies (Cell Signaling Technology, Danvers,Mass.).

Human Liver Histology

Human liver tissue samples were obtained, stained with hematoxylin andeosin (H&E) and imaged as described in Guarrera et al., Journal ofSurgical Research. 2011; 167(2):e365-e73.

Statistical Analysis

Results shown in text and graphs are mean±1 standard error. One-wayANOVA followed by Fisher's LSD post-hoc test was performed usingKaleidaGraph (Synergy Software, Reading, Pa.). Values of p<0.05 indicatestatistical significance.

Results

Macrosteatosis Induction in Primary Hepatocytes

Incubation of collagen sandwiched hepatocytes with steatosis-inducingmedium for 3 days (corresponding to 8 days post-seeding) induced amicrosteatotic appearance and increased the TG content ˜2.7 fold;consistent with prior studies (Berthiaume et al., Journal of SurgicalResearch. 2009; 152(1):54-60 and Nagrath et al. Metabolic Engineering.11(4-5):274-83) (FIG. 1B-C). Incubation with steatosis-inducing mediumfor an additional 3 days increased the TG content even further, reaching˜5 fold the level in lean controls (FIG. 1B-C). This level is consistentwith two in vivo models of liver macrosteatosis (ob/ob mouse and obeseZucker rat) where the intraheptic TG level is also ˜5 fold thecorresponding lean controls (Selzner et al., Journal of Hepatology.2006; 44(4):694-701, and Washizu et al., Tissue Engineering. 2000;6(5):497-504). The additional 3 days of steatosis induction also led toa dramatic increase in the lipid droplet size distribution (FIG. 1C).The number of lipid macrodroplets (cross-sectional surface area >350um², defining a macrosteatotic lipid droplet (FIG. 6) increased 20-foldcompared to the 3-day fatted cells (FIG. 1D). The 6-day fatted cellsexhibited morphological characteristics found in macrosteatotic humanlivers, notably large intracellular lipid droplets displacing thenucleus to the cell periphery, as shown in FIGS. 1B and E. In addition,lipid droplet cross sectional surface area was comparable to that inmacrosteatotic human livers (FIG. 1B, E). Hepatocyte viability was85-90% after the 3-day and 6-day fattening protocols and was notsignificantly different from lean controls (Table 1A).

TABLE 1 Hepatocyte Viability. Percent viable hepatocytes of theexperimental conditions at the relevant experimental days (based onimages obtained from the EtHD-1 viability assay; sample viabilitystaining is shown in FIG. 5). Data shown are means ± S.E. N = 5. Nostatistically significant change between treated hepatocytes andcorresponding lean controls on the same experimental day. A Day 8 Day 11Lean Hepatocytes 89.3% ± 1.0 88.5% ± 0.8 Steatosis induced Hepatocytes85.4% ± 1.8 86.4% ± 2.0 B Day 13 Day 15 Lean Hepatocytes 88.6% ± 1.285.6% ± 2.9 Macrosteatosis 82.0% ± 2.1 76.2% ± 2.5 Macrosteatosis 83.5%± 1.9 74.0% ± 3.8

Reduction of Macrosteatosis in Primary Hepatocytes

Using the 6-day macrosteatotic hepatocytes, the effect of macrosteatosisreduction on viability and liver-specific function was assessed. Toaccelerate macrosteatosis reduction, SRS previously shown to acceleratesteatosis reduction in microsteatotic cultured hepatocytes, wereutilized (Nagrath et al. Metabolic Engineering. 11(4-5):274-83). NSRS orSRS-containing medium was added to the macrosteatotic hepatocytes for 2days. When SRS medium was used, macrosteatosis reduction was accelerated˜4-fold compared to NSRS medium to yield ˜80% reduction within 2 days(FIG. 2A-B). Interestingly, both media compositions were equallyeffective in reducing TG content by ˜30% during that time frame (FIG.2C).

The size of individual lipid droplets in macrosteatotic hepatocytecultures during the first 36 h macrosteatosis reduction period decreasedlinearly as a function of time, while the nucleus returned towards thecenter of the hepatocyte (FIGS. 7A-B).

After 2 days in NSRS or SRS medium, all cells were switched to NSRSmedium for an additional 2 days (days 13-15 post-seeding). At the end ofthis observation period, regardless of the medium used to reducemacrosteatosis for the first 2 days, the hepatocytes exhibited steatosislevels comparable to control lean hepatocyte cultures (FIG. 2D). Thus,while macrosteatotic hepatocytes can eventually exhibit lean-like lipiddroplet distribution, SRS medium provides accelerated steatosisreduction during the first few hours.

Viability and Function Assessment

As indicated in FIG. 3A, macrosteatosis reduction was uneven among thehepatocytes. Viability assessment revealed that reduction occurred onlyin the viable population (EtHD-1 negative), suggesting that activemetabolism was critical. Nevertheless, during the first 2 macrosteatosisreduction days and the subsequent 2 days in NSRS medium, both SRS andNSRS medium treatments maintained hepatocyte viability similar to leancontrols on the same experimental day (Table 1B).

Next, cell function was assessed with liver-specific hepatocyte markerscommonly used to assess the function of hepatocyte cultures and liversin transplantation settings including albumin and urea secretion rates(FIG. 3B-D), as well as bile canalicular morphology and function (FIG.4) (Taneja et al., Transplantation. 1998; 65(2):167-72; Nakamuta et al.,Transplantation. 2005 Sep. 15; 80(5):608-12; Guarrera et al., Journal ofSurgical Research. 2011; 167(2):e365-e73; Jamieson et al.,Transplantation. 2011; 92(3):289-95; Ninomiya et al., Transplantation.2004; 77(3):373-9; Tuschl et al., Chemico-Biological Interactions. 2009;181(1):124-37; and Washizu et al., Tissue Engineering. 2000;6(5):497-504). Microsteatotic hepatocytes maintained the albuminsecretion rate and exhibited only a 1.7-fold decrease in urea secretionrates compared to lean hepatocyte cultures. On the other hand,macrosteatotic hepatocytes exhibited 1.5 fold lower albumin secretionlevels and a 5-fold reduction in urea secretion levels compared to leanhepatocyte cultures (FIG. 3B-DC). Thus, macrosteatosis induction for 6days had a more deleterious effect on hepatocyte function thanmicrosteatosis induction for 3 days.

In macrosteatosis induction and reversal cycled hepatocytes, albuminsecretion remained flat until the end of the observation time (day 15post-seeding) below lean hepatocyte control levels and did not recover.In this respect, macrosteatotic reduced hepatocytes, treated with SRS orNSRS, behaved similarly (FIG. 3B). On the other hand, the urea secretionrate at the end of the observation period returned to ˜50% of the leanhepatocyte culture levels in hepatocytes initially treated with NSRS andto ˜100% in hepatocytes initially treated with SRS medium (FIG. 3C).During the last 2 days, all the cultures received NSRS medium.Therefore, the improved urea secretion recovery rate in hepatocytesinitially treated with SRS medium at 15 days post-seeding can beattributed to the long-term effects of SRS treatment, such as theimproved macrosteatosis reduction (FIG. 3C).

In lean hepatocyte cultures, bile canaliculi and the accumulation offluorescent carboxy-DCFDA were easily visualized, suggesting afunctional bile canalicular network (FIG. 4A-B), consistent with similarstudies (Tuschl et al., Chemico-Biological Interactions. 2009;181(1):124-37). In macrosteatotic hepatocytes, neither fluorescentcarboxy-DCFDA nor the bile canalicular structure was observed (FIG.4A-B). Because carboxy-DCFDA requires cleavage by intracellularesterases to fluoresce and eventually be transported into the bile,assays were carried out to investigate whether esterase activity wasaffected by macrosteatosis. Similar cultures were incubated withcalcein-AM, which requires the same esterases to fluorescently label thecytoplasm. Calcein staining was similar in lean and macrosteatotichepatocytes, suggesting no impairment in esterase activity (FIG. 4C).Thus, the lack of carboxy-DCFDA accumulation in macrosteatotichepatocytes most likely reflects a disruption of the bile canalicularfunction.

Immediately following macrosteatosis reduction (13 days post-seeding),the bile canalicular morphology recovered completely to levels similarto lean controls, but only in hepatocytes treated with SRS medium (FIG.4A). Bile canalicular function partially recovered after macrosteatosisreduction using NSRS medium, while it appeared nearly complete andcomparable to lean controls after using the SRS medium (15 dayspost-seeding) (FIG. 4B). Similarly, the recovery in bile canalicularfunction was not attributed to levels of esterase activity (FIG. 4D).

In the assays, it was found that albumin secretion does not fullyrecover even if the culture time was extended. It is possible that theextent of defatting is not sufficiently complete to recover thisparticular function (since the data show that triglyceride content wasstill ˜68% of macrosteatotic levels, thus remaining well above the“lean” baseline). In contrast, macrosteatosis had no effect on levels ofhepatocyte nuclear factor 1-α, a transcription factor involved in theexpression of many liver-specific genes, arguing against ade-differentiation of the cells due to excessive fat accumulation(Pontoglio et al. Cell 1996; 84:575-585).

The above data show that hepatocytes induced for macrosteatosis after 6days exhibited lower albumin and urea secretion rates than those inducedfor microsteatosis after 3 days. This may be due to the direct effect ofthe increased lipid storage on these functions or to the difference inexposure times to FFA-rich medium. It is worth noting that in theclinic, albumin and urea blood data suggest that macrosteatotic patientsare asymptomatic, although they exhibit elevated liver enzymes. Thesetests, however, do not equate the hepatocyte production rates, as in theabove-described system, since blood levels are also affected byclearance mechanisms.

To explore the insulin sensitivity of the hepatocyte cultures, theresponse to insulin withdrawal followed by induction was investigated. Asmall increase in phosphorylated AKT could be seen after return toinsulin-rich medium in both in lean and macrosteatotic cultures;however, the high baseline in phosphorylated AKT limited the ability tostudy insulin responsiveness in this model.

More specifically, Western blot analysis was carried out to examine AKTand phosphorylated AKT (at Thr 308). As shown in FIG. 10, each lane wasloaded with intracellular protein extracted from 3 million hepatocytes.The blot was probed with antibodies for total AKT as well as forphosphorylated AKT. Data shown are representative of two separateexperiments that yielded similar results. A small increase inphosphorylated AKT could be seen after return to insulin rich medium inboth in lean and macrosteatotic cultures. It is noteworthy thathepatocytes that remained in insulin-free medium for 4 hours exhibited arelatively high baseline of phosphorylated AKT in both lean andmacrosteatotic conditions, suggesting a fairly persistent activation ofthe insulin pathway. In contrast, freshly isolated and seededhepatocytes that were never exposed to insulin rich media exhibitedlittle phosphorylated AKT, while 30 minutes of insulin exposuresignificantly increased those levels, reaching an intensity comparableto the long-term cultures (both lean and macrosteatotic).

The foregoing examples and description of the preferred embodimentsshould be taken as illustrating, rather than as limiting the presentinvention as defined by the claims. As will be readily appreciated,numerous variations and combinations of the features set forth above canbe utilized without departing from the present invention as set forth inthe claims. Such variations are not regarded as a departure from thescope of the invention, and all such variations are intended to beincluded within the scope of the following claims. All references citedherein are incorporated herein in their entireties.

What is claimed is:
 1. A method of producing a population of culturedmacrosteatotic hepatocytes, comprising: providing a population ofhepatocytes; culturing said population of hepatocytes on a collagenmatrix for a first period of time; distributing a collagen gel solutionover the hepatocytes to create a collagen sandwich configuration so thatthe hepatocytes are in the middle of the collagen sandwichconfiguration; culturing the hepatocytes in the collagen sandwichconfiguration for a second period of time; culturing the hepatocytes ina steatosis inducing medium for a third period of time to obtain saidpopulation of cultured macrosteatotic hepatocytes, wherein the thirdperiod of time is about 3-9 days.
 2. The method of claim 1, wherein thefirst period of time is about 12 hours to 48 hours, about 18 hours to 30hours, or about 24 hours.
 3. The method of claim 1, wherein the secondperiod of time is about 2 days to 3 weeks, about 5-7 days, or about 6days.
 4. The method of claim 1, wherein the third period of time isabout 5-7 days, or about 6 days.
 5. The method of claim 1, wherein thesteatosis inducing medium contains one or more agent selected from thegroup consisting of a fatty acid, bovine serum albumin (BSA), insulin,glucose, and heparinized plasma.
 6. The method of claim 5, wherein thefatty acid is oleic acid, linoleic acid, or palmatic acid.
 7. The methodof claim 1, wherein the population of hepatocytes is from a non-humanmammal.
 8. The method of claim 7, wherein the non-human mammal is arodent.
 9. The method of claim 1, wherein the population of hepatocytesis from a human.
 10. The method of claim 1, wherein the macrosteatotichepatocytes contain one or more large lipid droplets.